The detection of specific binding between biomolecules, such as antigens, antibodies, the complementary strands of nucleic acid, and ligands and receptors is a key experimental assay in biological research. Given the complexity of biological systems, there is a need for assays that assess the binding of many different potential binding pairs. Assays that provide for rapid, parallel, compact, and inexpensive testing for binding between macromolecules are desired and accelerate discovery of new therapeutic drugs.
High throughput automation has been developed for biomedical and pharmaceutical research. In this area, instruments are designed to handle microtiter plates. These plates conform to standards developed by the Society for Biomolecular Screening and were published in conjunction with the American National Standards Institute (SBS Standards), which are conformed to by most, if not all, manufacturers of microtiter plates. Wells in these plates are designed with standard spacing, for example, a 96-well plate has twelve columns and eight rows with 9 mm spacing between the centers of adjacent wells. A 384-well plate has twenty-four columns and sixteen rows with 4.5 mm spacing between the centers of adjacent wells. A 1536-well plate has forty-eight columns and thirty-two rows with 2.25 mm spacing between the centers of adjacent wells. Pipetting and plate-washing robotic instruments have been designed to handle plates conforming to this standard. Optical instrumentation has also been designed around the SBS microtiter plate standards.
Arrays are commonly used to process assays based on specific binding of biological molecules, e.g., antibodies to antigens or the complementary strands of nucleic acid to each other or ligands to receptors. One simple mode of using arrays typically entails coating the bottom surface of each well of an array with a molecule, (e.g., an antibody) to provide an immobilized molecule and then adding different fluid samples which may or may not contain a molecule that binds to the immobilized molecule (e.g., an antigen specifically bound by the antibody) to each well. After allowing for binding, the fluid sample is removed, and the well is washed to remove unbound material. Binding or lack of binding is then detected using well established methods. Alternatively, different samples may be used to coat different wells, and a common fluid sample may then be added to all the wells, followed by detection of binding. Each well in either mode is used to produce a single data point related to the binding of, for instance, an antibody to an antigen. Use of microtiter plates having, for instance, 1536 wells allow many assays to be done in parallel, but owing to the small size of each well and the complexity of the liquid handling required, it is difficult for these assays to be performed reliably by hand, and robotic systems are almost always used.
Arrays comprised of such biomolecules as DNA, oligonucleotides, oligopeptides, polypeptides and proteins have become standard tools for monitoring changes in gene expression, and for detecting and measuring specific binding between biomolecules or between biomolecules and other compounds. This technique has found rapidly growing use in many molecular biology, biochemistry, immunology, pharmacology and functional genomics or proteomics laboratories. In array based assays hundreds to tens of thousands of probes are attached to a substrate in an array format, and sample solutions are incubated on these arrays. Many samples are often tested for binding to many different biomolecules which have been immobilized to sites within the array. These assays require efficient processing of samples and analysis of results. Automation, particularly robotic automation, of these processes is particularly valuable.
A system that allows multiple fluid samples to be tested for binding to multiple immobilized materials which would be amenable to manual handling while being compact and easy to manufacture is desired. A system that also facilitates the use of robotic systems when available and appropriate is also desired. One approach widely used to assemble multiple sites of immobilized molecules involves spotting of small volumes on a flat surface such as glass to produce small regions of bound molecules. This spotting process requires precise targeting during the spotting process and the volume that can be spotted in a defined area is limited.
Currently, DNA or protein microarrays are commonly made using glass microscope slides. In a typical process, a robotic “spotter” is used to deposit small amounts of fluids containing the probes onto a glass slide to form an array. Approximately tens of thousands of spots can be arrayed onto a standard glass slide. The DNA or protein is maintained at its site of application solely by its binding to the surface, and the spatial definition and location of the binding is dependent on the precision of the spotting process. No physical barrier such as a wall is used to define the boundary of the spot or its location. Next, a solution of labeled targets is applied on the slide, typically by hand. After the slide is then placed in an incubator/mixer for a time effective for binding to occur, often hours. The fluorescent signals from the bound targets are then imaged using a laser and a photomultiplier tube (e.g. a laser scanner). This method, however, suffers from low throughput, poor data quality and poor reproducibility.
An alternative to using glass slides would be to use existing molded polymer microtiter plates, where arrays are printed on the bottom of the wells. The microtiter plate is a well-known tool; the attachment of biomolecules or other molecules within the microwell either as single elements (e.g., ELISA plates) or in the form of arrays for example those described in International Application publication number WO 98/29736, which is hereby incorporated by reference, is also well known. The reading of arrays, however, is difficult in conventional microwells without the use of expensive equipment. There are also limitations in terms of field of focus and uniform lighting of the bottom of the well using conventional microtiter plates. In addition, the non-symmetrical design of these plates (solid bottom with open topped wells) results in molded parts that warp due to differential thermal expansion. In recent years, methodologies have been developed to print multiple spots onto the bottoms of the microtiter plates (e.g. glass bottom plates) using pin type arrayers. Special imaging instruments are also now available to image this type of in-well microarray. Means to construct arrays on such a scale that do not require high precision and which permit more forgiving manufacturing tolerances than are required to make the above arrays would be desirable. Arrays that allow for use of a larger volume of solution per locus constructed than is feasible with the above spotting methods would be desirable. Arrays of this scale are commonly called microarrays.
In a typical microtiter plate the surface area available for analyte binding for practical purposes is limited to the bottom surface plus the side walls up to at most halfway to the top of the well. A system that maximizes the surface area to which the immobilized binding partner is bound while allowing for effective washing of unbound material in the fluid sample without increasing the overall scale of the system is also desired.
Traditionally, arrays are processed by washing them with a single sample at a time, e.g., serum taken from a single patient. With the further extension of arraying technology, it would be desirable to study the interaction of many different samples with a given array of materials. For example, one may want to screen thousands of different serum samples from patients with an array of 96 different antigens. Alternatively, one may wish to screen thousands of different small organic compounds for their ability to disrupt or prevent protein-protein interactions in an array of 96 different pairs of proteins. To do this, it would be valuable to use the current instrumentation for preparing and scanning arrays in combination with the current instrumentation for processing samples in microtiter plates.
Given the wide acceptance of liquid handling robotics designed to use microtiter plates that conform to an SBS Standard, there is a need in the art for a high-throughput system that allows arrays which conform to an SBS Standard microtiter plate to be processed using robotics, and for such a system that produces high quality data.